The iPSC perspective on schizophrenia

Over a decade of schizophrenia research using human induced pluripotent stem cell (iPSC)-derived neural models has provided substantial data describing neurobiological characteristics of the disorder in vitro. Simultaneously, translation of the results into general mechanistic concepts underlying schizophrenia pathophysiology has been trailing behind. Given that modeling brain function using cell cultures is challenging, the gap between the in vitro models and schizophrenia as a clinical disorder has remained wide. In this review, we highlight reproducible findings and emerging trends in recent schizophrenia-related iPSC studies. We illuminate the relevance of the results in the context of human brain development, with a focus on processes coinciding with critical developmental periods for schizophrenia.Peer reviewe

Genetic contribution to the aggregation of schizophrenia and bipolar disorder in multiplex consanguineous Pakistani pedigrees

La schizophrénie (SCZ) et le trouble bipolaire (TB) sont des troubles mentaux graves qui présentent tous deux des symptômes affectifs et psychotiques. La SCZ est un trouble psychotique primaire caractérisé par des symptômes d’idées délirantes et d’hallucinations. Le TB est principalement un trouble de l'humeur primaire défini des périodes de manie et de dépression. En 2010, ces troubles contribuaient respectivement à 7,4% et 7,0% de la charge mondiale de morbidité. La prévalence élevée (~ 0,4% pour la SCZ et ~ 2,4% pour le TB) et la forte héritabilité estimée (~ 80%) suggèrent toutes deux une forte influence génétique. Les données disponibles démontrent qu'il existe des chevauchements génétiques entre les deux conditions, mais également des composantes génétiques spécifiques à chaque maladie. Au cours de la dernière décennie, des études d’association pan-génomiques ont identifié des centaines de loci génétiques associés à ces maladies. De plus, d’autres méthodes ont permis de mettre en relief la contribution d’autres types de variations génétiques comme les rares variations du nombre de copies (CNV), de rares polymorphismes de nucléotide simple (SNV) et des mutations de novo (MDN). Bien que notre connaissance de l'architecture génétique de ces conditions est en progression, une grande partie de l'héritabilité demeure toujours non résolue et inexpliquée. Une longue histoire de faible mélange génétique combiné à la pratique répandue de mariages consanguins (50% des unions sont consanguines) rend les familles pakistanaises prometteuses pour des études génétiques médicales basées sur la population. Des études épidémiologiques ont démontré que la consanguinité est associée à un risque accru de nombreux traits. L’étude de familles a largement été appliquée dans la cartographie génétique des caractères mendéliens et complexes. Cependant, peu d’études ont eu recours à de grandes familles consanguines multiplexes pour étudier en profondeur le rôle de la consanguinité dans les troubles neuropsychiatriques tels que la SCZ et le TB. Les CNVs ont été impliquées dans la SCZ et le TB depuis la découverte des délétions 22q11.2. Malgré que ces derniers soient rares dans la population, ils contribuent de manière significative au risque. Des études d'association de CNV ont révélé un enrichissement de délétions et de duplications rares et un taux plus élevé de CNV de novo dans les cas relatifs aux témoins. De plus, le séquençage du génome de familles SCZ a révélé une charge accrue de rares CNVs exonics chez les sujets SCZ ainsi que de l'hétérogénéité génétique. L'utilisation de grandes familles de multiplexes pourrait être statistiquement puissante pour étudier le rôle des CNVs co-ségrégant avec la maladie et éventuellement pathogènes. Afin de mieux comprendre l'hétérogénéité génétique et résoudre l’héritabilité manquante de ces deux troubles mentaux, nous avons utilisé du génotypage et du séquençage de l'exome afin d’examiner le profil génétique de grandes généalogies consanguines multiplexes d’origine Parkistanaise. Chacune de ces familles comportait plus de dix membres affectés par la SCZ ou le TB. Dans cette thèse, nous caractérisons la population d’origine, ce qui comprend le mélange génétique et la consanguinité récente de notre cohorte. Nous avons testé si le niveau de consanguinité était associé au phénotype binaire et à ses dimensions sous-phénotypiques. Nous avons également inclus un grand ensemble de données de populations contrôles externes et appariées afin de calculer et comparer le coefficient de consanguinité. Notre approche, qui comprenait une analyse de liaison, une cartographie de l’auto-zygosité, la détection de cycles homozygotie et une analyse de ségrégation de variantes homozygotes délétères rares, nous a conduit à rejeter l’hypothèse d’un modèle de transmission récessif sur ces familles (malgré leur forte consanguinité). Par la suite, nous avons examiné si des CNVs co-ségrégaient avec le phénotype dans certaines familles. Cette étude comportait plusieurs étapes: 1 - une comparaison systématique entre différents algorithmes de détection de CNVs. 2 - une validation croisée de vrais CNVs ou de faux positifs par des approches in silico ou expérimentales, 3 - le développement d’un logiciel de ségrégation et d'annotation. Cette étude met de l’avant à la fois les avancées méthodologiques et les limites de l’exploration des CNVs. Au final, aucun des CNVs identifiés ne semblent contribuer à la variance génétique de la SCZ et du TB des familles examinées dans cette étude. Les résultats présentés dans cette thèse étayent une hypothèse alternative qui impliquerait des interactions polygéniques entre à la fois des variants rares et des variants communs.Schizophrenia (SCZ) and bipolar disorder (BP) are two major psychiatric disorders. SCZ is a primary psychotic disorder that typically involves symptoms of delusions and hallucinations, by comparison BP is a mood disorder engaging mania and depression but it can also involve psychosis. A 2010 estimation of these disorders highlighted that they respectively contributed to ~7.4% and ~7.0% of the global burden of disease. The high prevalence (~0.4% for SCZ and ~2.4% for BP) and estimated heritability (~80%) suggest a strong genetic influence. Evidence shows that there are some genetic overlaps between the two conditions but also disorder-independent genetic components. Over the past decade, genome-wide association studies (GWAS) identified hundreds of SCZ and BP loci, and other approaches identified various forms of potential genetic risk factors, for instance rare copy number variants (CNVs), rare single nucleotide variants (SNVs) and de novo mutations (DNMs). While our knowledge of the genetic architecture of these conditions grow, a large of portion of the genetic heritability of each disorder still remains unexplained. The combination of a long history of genetic admixture, and the tradition of consanguineous marriages (50% of unions are consanguineous), makes Pakistani families promising for population based medical genetics studies. Consanguinity has previously been associated with an increased risk of numerous traits in epidemiological studies. Family-based designs have been widely applied in the genetic mapping of Mendelian and complex traits. However, few studies have used large multiplex consanguineous families to thoroughly investigate the role of consanguinity in neuropsychiatric disorders such as SCZ and BP. CNVs have been implicated in SCZ and BP since the discovery of 22q11.2 deletions, however, most of them are rare in the population but contribute significantly to the risk. Association studies of CNVs found enrichment of rare deletions and duplications, and a higher rate of de novo CNVs in cases relative to controls. Whole-genome sequencing of multiplex SCZ families reported increased burden of rare, exonic CNV in SCZ probands and genetic heterogeneity. Using large multiplex families could be statistically powerful to investigate the role of segregating, and possibly pathogenic, CNVs. In order to better understand the genetic heterogeneity and look for missing heritability of these two common disorders in Pakistani families, we used SNP genotyping and whole-exome sequencing to examine the genetic profile of ten large multiplex consanguineous pedigrees; each of these families involved more than ten members affected by SCZ or BP. In this thesis, we characterized the population background which includes admixture and recent inbreeding of our cohort. We tested if the inbreeding level was associated with the binary phenotype and its subphenotype dimensions. We also included large external dataset of matched population control individuals to compute and compare the inbreeding coefficient. Our approach, which included linkage analysis, autozygosity mapping, runs of homozygosity (ROH) and rare deleterious homozygous variants segregation analysis, led us to reject the hypothesis of a recessive inheritance model across these families (despite of their high inbreeding). We subsequently looked if any CNV segregated across some of the families. This examination involved multiple steps: 1 - a systematic comparison of a range of CNV detection algorithms currently available through different platforms, 2 - a cross validation of true and false positive CNV calls through the use of in silico or experimental approaches, 3 - the development of our own segregation and annotation software. This effort both emphasized the methodological advances and limitations of CNV studies. In the end, none of the potentially pathogenic CNV identified appeared to account for the genetic variance of SCZ and BP observed in the families examined here. The results presented in this thesis provide support for an alternate hypothesis that would involve a polygenic pattern where both rare variants and common variants would be at play

Association of a Chromosomal Susceptibility Locus to Bipolar Affective Disorder (BPAD): A case-control study in Kashmir

The etiology of bipolar disorder (BPAD) is still unknown, but family, twin, and adoption studies strongly suggest the involvement of genetic risk factors. Linkage studies have revealed a number of loci to be linked with BPAD. Of those, several investigators confirmed 18p11 as one of the susceptibility loci for BPAD. Thus, chromosome 18p11 is one of the targets of the genetic association study of BPAD. The aim of our study was to investigate whether the tandem repeat polymorphism in D18S452 microsatellite marker at locus 18p11.2 is a risk factor for the development of BPAD in Kashmiri population. The repeat polymorphism in D18S452 was evaluated in 74 patients with BPAD and 74 control (age, sex and region matched) individuals. The repeat polymorphism was evaluated by PCR analysis of DNA obtained from the blood of the subjects. We observed that the tandem repeat (300bp*) allele frequency was found to be 1.35 % in the controls and 8.108% in cases. The tandem repeat (250bp*) allele frequency was found to be in 91.89% in cases and 98.65% in controls. The 252bp/252bp genotype was found to be present in 89.18% of the cases and 98.64% of the controls, the 300bp/300bp genotype in 5.40% of cases and 1.35% of controls and the 252bp/300bp variant in 5.40% of the cases and none among the controls. It was observed that although the proportion of patients homozygous for the tandem repeat (300bp/300bp) was higher in cases than in controls, the difference was not statistically significant when using 252bp/252bp genotype as a reference (OR= 4.4242; 95% CI, 0.4822-40.5924); P = 0.1529). However, it was observed that the frequency of the heterozygous genotype (252bp/300bp) when compared with 252bp/252bp showed statistical significance (OR=8.0603; 95% CI, 1.1112-58.4646; P = 0.0383). Chromosome 18 harbors many candidate genes that are involved in the pathophysiology of BPAD. It may be possible that this marker is directly or indirectly involved in the regulation of neighboring genes. It is also possible that this locus may be in linkage disequilibrium with other genes. Although, this is the first study reporting the association of the marker D18S452 in heterozygous condition (252bp/300bp) with BPAD. Yet, it would be too early to associate this genotype with the predisposition to BPAD. Therefore, further studies with larger sample size should be carried to validate the result, taking into account the various disease phenotypes, endophenotypes and environmental conditions

Early SHH-Dependent Telencephalic Patterning Disruptions in Tourette Syndrome

Ventral telencephalic development gives rise to the basal ganglia, a subpallial brain region responsible for motor function and coordination. This brain region is implicated in many movement disorders, including Tourette Syndrome (TS). TS is a heterogenous neurodevelopmental disorder and its etiopathophysiology is unknown. To date, TS has been investigated in animal models and postnatal human subjects, but early development of this disorder has not been studied. Previous work in adult TS post mortem basal ganglia tissue has shown a reduction in striatal interneurons, which serve to largely regulate striatal output. However, possible mechanisms for this neuronal loss and whether or not these findings originate in early development are poorly understood. This study examines TS etiology by modeling basal ganglia development in tridimensional human induced pluripotent stem cell-derived neural organoids. Basal ganglia organoids were generated from and compared across healthy unaffected control individuals and adult unremitting TS patients. We found early telencephalic patterning disruptions in TS-derived basal ganglia organoids, showing a preference for dorsal-posterior specification instead of the expected ventral-anterior commitment seen in healthy control-derived organoids. The aberrant fate shift in the TS-derived basal ganglia organoids was seen at both RNA and protein levels, confirmed across three separate assays, with consistency across three distinct time points. Transcriptome analyses in the organoids further identified categories of neuronal deficits that show overlap with a manually curated list of differentially expressed genes uncovered by transcriptome analyses at the post mortem level, reiterating the relevance of the bioassay utilized in this study. This work also investigated a potential mechanism for the early developmental phenotypes observed in the TS organoids. We found significant alterations in sonic hedgehog (SHH) signaling components at both RNA and protein levels that are essential for distinguishing dorsal-ventral patterning in the human brain. Additionally, transcriptome analyses reveal a potential role for cilia, the cellular protrusions that facilitate SHH signal transduction. We found disruptions in genes that are required for cilia formation and function in the TS basal ganglia organoids that were absent from the healthy controls. This study leads an early developmental examination of TS in humans and offers a bioassay applicable to modeling basal ganglia-related disorders. These results reveal new biomarkers of interest in TS etiology and describe a new implication for SHH signaling. These results indicate that TS patients may exhibit altered telencephalic development, which yields deficits in neurons that ultimately populate the basal ganglia and regulate optimal circuitry function

Understanding psychiatric risk from DLG2 haploinsufficiency CNVs through the phenotyping of a Dlg2+/- rat model

Copy number variants at the 11q14.1 locus are associated with multiple psychiatric conditions (e.g. Kirov et al., 2012; Gao et al., 2018). The candidate gene within this CNV, which is either completely deleted or duplicated, is DLG2 (coding PSD-93). PSD-93, a synaptic scaffold protein, stabilises effectors in the postsynaptic density of excitatory synapses. If PSD-93 content is altered key synaptic processes may be disrupted. Phenotyping a rat model heterozygous for Dlg2 (+/-), which models the deletion CNV in patients, can help isolate endophenotypes with cross-disorder relevance. At the protein level Dlg2+/- rats show a decrease in PSD-93 without changes to PSD-95 or NR1 expression. Ex-vivo structural MRI scans were analysed for white matter abnormalities and differences in grey matter volume; however no genotype effects were seen. Behavioural phenotyping was conducted using assays relevant to symptom domains seen in 11q14.1 deletion syndromes including anxiety, social behaviour, PCP-hyperlocomotion, and sensorimotor gating. Memory and learning in a battery of object recognition tasks and water-maze reference memory were assessed as an index of cognitive ability. Hedonic responses to stimuli, appetitive conditioning, and motivation to work for reward were assessed to capture reward processing. Finally, a novel paradigm to assess the propensity towards hallucinations and delusions in rodents was conducted. Dlg2+/- rats performed as wild-types on almost all measured domains and tasks. In many cases this presents a departure from findings with Dlg2 homozygous (-/-) knockout mice who demonstrate increased anxiety and deficient social behaviour. With the caveats of drawing conclusions across species and experiments, this highlights the importance of using the most clinically valid (i.e. heterozygous) models when characterising the effects of CNVs. Dlg2+/- rats showed a potentiated and sustained locomotor response to the psychostimulant PCP, showing that although its effects are subtle, deleting one copy of Dlg2 in a rat model does result in a compromised system with a possible psychosis susceptibility

Omics-based predictive and causative modeling of neurobehavioral traits

Neurobehavioral disorders can be phenotypically and genetically complex, and often diagnosed through observational study or subjective assessment alone. Certain neurobehavioral phenotypes, such as those caused by circadian rhythm related behavior, are biochemically well characterized, others, though, do not have yet a well understood genetic aetiology. Furthermore, circadian biology and psychological disorders are often intertwined. To advance our understanding of neurobehavioral trait/gene relationships, I first built a machine learning model that encompasses mouse transcriptomics to predict genes involved in circadian rhythms. Next, I used genome wide association studies to model the causal influence of genetic exposure in humans to an evening chronotype on several mental health and social support traits, from depression to group religious participation. To more accurately model how neurobehaviors relate to one another, I mined psychological assessment instruments to build a species-agnostic psychological neurobehavior ontology encompassing autism and schizophrenia phenotypes. I, then, tested the utility of this ontology in clustering children on the autism spectrum based on phenotypic profiles. Lastly, I annotated genes to behaviors identified among subgroups through genome wide association studies applied to phenotype profiles. This allowed for the gene prioritization of circadian related experimentation results and the discovery of new, potentially, casual relationships between chronotype and neurobehavioral traits. Finally, the semantic representation of schizophrenia endophenotypes in a consistent, ontology framework catered its application for the identification of novel gene-trait associations in humans. These contributions provide new knowledge to the scientific community of the potential novel circadian functions for known genes, of the likely causal influence of chronotype on social and mental health, provide novel robust ways of modeling the complex phenotype of autism and schizophrenia patients, while annotating neurologically active genes to new behavioral traits for the first time